Platelet activation is a principal determinant of the arterial thrombotic response. The endothelial cell plays a key role in limiting platelet activation by producing the platelet inhibitors, prostacyclin and nitric oxide. Extracellular antioxidant enzymes, including extracellular superoxide dismutase and plasma glutathione peroxidase, limit the inactivation of endothelial nitric oxide by reactive oxygen species. Plasma glutathione peroxidase (GPx-3) is a principal antioxidant enzyme in the vascular compartment. This extracellular peroxidase is a selenocysteine-containing enzyme that reduces hydrogen and lipid peroxides to their corresponding alcohols, and does so using either glutathione or thioredoxin as reducing equivalents. We have previously demonstrated that GPx-3 potentiates the inhibition of platelet function by nitric oxide by limiting the availability of lipid peroxides (and derivative peroxyl radicals), and that a deficiency of GPx-3 can lead to an arterial thrombotic diathesis. Aside from these initial observations, little is known about the vascular biology of GPx-3 or the molecular basis of its protective antioxidant and antithrombotic benefits in vivo. In this competing renewal application, we propose to focus on the molecular biology and (patho)physiology of GPx-3 using a combination of molecular, cellular, and genetic approaches. We hypothesize that a deficiency of GPx-3 increases vascular oxidant stress, decreases bioavailable nitric oxide, and promotes platelet-dependent arterial thrombus formation. To test this hypothesis, we will define the molecular determinants of GPx-3 gene and protein expression, assess the effects of GPx-3 over expression or suppression on (extra)cellular oxidant stress in vascular cells and platelets, and characterize the consequences of GPx-3 over expression or deficiency on vascular function and platelet activation in genetic mouse models. With these studies, we hope to understand better the role of this important extracellular antioxidant enzyme in preventing platelet activation and thrombosis, and in potentiating nitric oxide bioactivity in the vasculature.